Functional characterization of CYP3A4.16: Catalytic activities toward midazolam and carbamazepine

Abstract

1. To assess the substrate-dependent effects of the low-activity allele of human CYP3A4, CYP3A4*16 (Thr185Ser), a recombinant wild-type (CYP3A4.1) or variant (CYP3A4.16) protein was co-expressed with human NADPH-P450 reductase in Sf21 insect cells using a baculovirus-insect cell system. 2. The holo-CYP3A4 protein level of CYP3A4.16 in insect microsomes was slightly higher than that of CYP3A4.1, while no difference in total (apo- and holo-) CYP3A4 protein levels was observed between them. 3. When midazolam was used as a substrate, K(m) and V(max) for 1'-hydroxylation in CYP3A4.16 were significantly higher and lower, respectively, than those in the wild-type, resulting in a 50% decrease in intrinsic clearance (V(max)/K(m)) of the variant. In contrast, intrinsic clearance for 4-hydroxylation of the variant was decreased by 30% due to a significant increase in K(m) without a difference in V(max). 4. Both the wild-type and variant exhibited sigmoidal kinetic profiles for carbamazepine 10,11-epoxide formation. When the modified two-site equation was applied for the analysis of kinetic parameters, K(m2) and V(max2) of CYP3A4.16 were approximately two times higher and lower than those of the wild-type, resulting in a 74% decrease in intrinsic clearance. 5. These results demonstrated that CYP3A4.16 shows the substrate-dependent altered kinetics compared with CYP3A4.1.

CYP3A4*16andCYP3A4*18Alleles Found in East Asians Exhibit Differential Catalytic Activities for Seven CYP3A4 Substrate Drugs

“…Insect cell microsomes coexpressing CYP3A4 (wild type or variants) and NADPH P450 reductase (OR) were prepared according to methods described previously (Maekawa et al, 2009). The cytochrome P450 content and OR activity in microsomes were measured (Phillips and Langdon, 1962;Omura and Sato, 1964), and Western blotting of CYP3A4 and OR was performed as described previously (Maekawa et al, 2009).…”

“…Catalytic activities for MDZ 1Ј-and 4-hydroxylations and CBZ 10,11-epoxide formation were measured as described previously (Maekawa et al, 2009), with slight modifications. For other substrates (ATV, PTX, DTX, IRN, and TFN), the incubation conditions were similar to those used for MDZ and CBZ.…”

“…CYP3A4.16 exhibited an approximately 50% reduction in intrinsic clearance (V max /K m ) for testosterone (TST) 6␤-hydroxylation activity in vitro (Murayama et al, 2002). We recently demonstrated the substratedependent altered kinetics of CYP3A4.16 for midazolam (MDZ) and carbamazepine (CBZ) (Maekawa et al, 2009). The intrinsic clearance for 1Ј-hydroxymidazolam (1Ј-OH-MDZ), 4-hydroxymidazolam (4-OH-MDZ), and CBZ 10,11-epoxide formation decreased by 50, 30, and 74%, respectively, compared with the wild type.…”

Current Approaches for Investigating and Predicting Cytochrome P450 3A4-Ligand Interactions

“…Positive cooperativity was also detected in the binding of aflatoxin B1 (n H of 2.3) [12], α-naphthoflavone (ANF; n H of 1.2–1.7) [11, 13], and Nile Red [NR] (n H of 1.6) [14]. Acetaminophen and midazolam bind to CYP3A4 with a negative cooperativity [15, 16], whereas progesterone and 7-benzyloxyquinoline (BQ) display both positive and negative cooperativity [10, 17]. Structures of these substrates are shown in Fig.…”

Pharmacogenetic potential biomarkers for carbamazepine adverse drug reactions and clinical response

“…Several genetic polymorphisms in CYP3A4 are known to affect its catalytic activity and to contribute in part to interindividual differences in the pharmacokinetics and pharmacodynamics of CYP3A4 substrate drugs. Maekawa et al, demonstrated the substrate dependent altered kinetics of CYP3A4*16 allele for midazolam and CBZ [62,63]. They also found lowered catalytic activities of the two alleles found in East Asians, CYP3A4*16 (Thr185Ser) and CYP3A4*18 (Leu293Pro), for metabolism of some drug substrates, including CBZ compared with wild type allele, CYP3A4*1 [63].…”